A non-oscillatory, millisecond-scale embedding of brain state provides insight into behavior.

Sleep and wake are understood to be slow, long-lasting processes that span the entire brain. Brain states correlate with many neurophysiological changes, yet the most robust and reliable signature of state is enriched in rhythms between 0.1 and 20 Hz. The possibility that the fundamental unit of brain state could be a reliable structure at the scale of milliseconds and microns has not been addressed due to the physical limits associated with oscillation-based definitions. Here, by analyzing high resolution neural activity recorded in 10 anatomically and functionally diverse regions of the murine brain over 24 h, we reveal a mechanistically distinct embedding of state in the brain. Sleep and wake states can be accurately classified from on the order of 100 to 101 ms of neuronal activity sampled from 100 µm of brain tissue. In contrast to canonical rhythms, this embedding persists above 1,000 Hz. This high frequency embedding is robust to substates and rapid events such as sharp wave ripples and cortical ON/OFF states. To ascertain whether such fast and local structure is meaningful, we leveraged our observation that individual circuits intermittently switch states independently of the rest of the brain. Brief state discontinuities in subsets of circuits correspond with brief behavioral discontinuities during both sleep and wake. Our results suggest that the fundamental unit of state in the brain is consistent with the spatial and temporal scale of neuronal computation, and that this resolution can contribute to an understanding of cognition and behavior.

Identification of activity-induced Egr3-dependent genes reveals genes associated with DNA damage response and schizophrenia.

Bioinformatics and network studies have identified the immediate early gene transcription factor early growth response 3 (EGR3) as a master regulator of genes differentially expressed in the brains of patients with neuropsychiatric illnesses ranging from schizophrenia and bipolar disorder to Alzheimer's disease. However, few studies have identified and validated Egr3-dependent genes in the mammalian brain. We have previously shown that Egr3 is required for stress-responsive behavior, memory, and hippocampal long-term depression in mice. To identify Egr3-dependent genes that may regulate these processes, we conducted an expression microarray on hippocampi from wildtype (WT) and Egr3-/- mice following electroconvulsive seizure (ECS), a stimulus that induces maximal expression of immediate early genes including Egr3. We identified 69 genes that were differentially expressed between WT and Egr3-/- mice one hour following ECS. Bioinformatic analyses showed that many of these are altered in, or associated with, schizophrenia, including Mef2c and Calb2. Enrichr pathway analysis revealed the GADD45 (growth arrest and DNA-damage-inducible) family (Gadd45b, Gadd45g) as a leading group of differentially expressed genes. Together with differentially expressed genes in the AP-1 transcription factor family genes (Fos, Fosb), and the centromere organization protein Cenpa, these results revealed that Egr3 is required for activity-dependent expression of genes involved in the DNA damage response. Our findings show that EGR3 is critical for the expression of genes that are mis-expressed in schizophrenia and reveal a novel requirement for EGR3 in the expression of genes involved in activity-induced DNA damage response.

Currently Unstable: Daily Ups and Downs in E-I Balance.

Balance between excitation and inhibition (E-I balance) in neural circuits is believed to be tightly regulated. To the contrary, in this issue of Neuron, Bridi et al. (2020) reveal that inverse oscillations of synaptic inhibition and excitation lead to peaks and valleys in E-I balance across the 24 h day.

The Immediate Early Gene Egr3 Is Required for Hippocampal Induction of Bdnf by Electroconvulsive Stimulation.

Early growth response 3 (Egr3) is an immediate early gene (IEG) that is regulated downstream of a cascade of genes associated with risk for psychiatric disorders, and dysfunction of Egr3 itself has been implicated in schizophrenia, bipolar disorder, and depression. As an activity-dependent transcription factor, EGR3 is poised to regulate the neuronal expression of target genes in response to environmental events. In the current study, we sought to identify a downstream target of EGR3 with the goal of further elucidating genes in this biological pathway relevant for psychiatric illness risk. We used electroconvulsive stimulation (ECS) to induce high-level expression of IEGs in the brain, and conducted expression microarray to identify genes differentially regulated in the hippocampus of Egr3-deficient (-/-) mice compared to their wildtype (WT) littermates. Our results replicated previous work showing that ECS induces high-level expression of the brain-derived neurotrophic factor (Bdnf) in the hippocampus of WT mice. However, we found that this induction is absent in Egr3-/- mice. Quantitative real-time PCR (qRT-PCR) validated the microarray results (performed in males) and replicated the findings in two separate cohorts of female mice. Follow-up studies of activity-dependent Bdnf exons demonstrated that ECS-induced expression of both exons IV and VI requires Egr3. In situ hybridization demonstrated high-level cellular expression of Bdnf in the hippocampal dentate gyrus following ECS in WT, but not Egr3-/-, mice. Bdnf promoter analysis revealed eight putative EGR3 binding sites in the Bdnf promoter, suggesting a mechanism through which EGR3 may directly regulate Bdnf gene expression. These findings do not appear to result from a defect in the development of hippocampal neurons in Egr3-/- mice, as cell counts in tissue sections stained with anti-NeuN antibodies, a neuron-specific marker, did not differ between Egr3-/- and WT mice. In addition, Sholl analysis and counts of dendritic spines in golgi-stained hippocampal sections revealed no difference in dendritic morphology or synaptic spine density in Egr3-/-, compared to WT, mice. These findings indicate that Egr3 is required for ECS-induced expression of Bdnf in the hippocampus and suggest that Bdnf may be a downstream gene in our previously identified biologically pathway for psychiatric illness susceptibility.

Positive Allosteric Modulation as a Potential Therapeutic Strategy in Anti-NMDA Receptor Encephalitis.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction.SIGNIFICANCE STATEMENT Anti-N-methyl-d-aspartate receptor encephalitis (NMDARE) is increasingly recognized as an important cause of sudden-onset psychosis and other neuropsychiatric symptoms. Current treatment leaves unmet medical need. Here we demonstrate cellular evidence that newly identified positive allosteric modulators of NMDAR function may be a viable therapeutic strategy.

Effects of a 5-HT1B Receptor Agonist on Locomotion and Reinstatement of Cocaine-Conditioned Place Preference after Abstinence from Repeated Injections in Mice.

5-HT1B receptors (5-HT1BRs) modulate behavioral effects of cocaine. Here we examined the effects of the 5-HT1BR agonist 5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine (CP94253) on spontaneous and cocaine-induced locomotion and on cocaine-primed reinstatement of conditioned place preference (CPP) in male mice given daily repeated injections of either saline or cocaine (15 mg/kg, IP) for 20 days. In the locomotor activity experiment, testing occurred both 1 and 20 days after the final injection. In the CPP experiment, mice underwent conditioning procedures while receiving the last of their daily injections, which were given either during or ≥2 h after CPP procedures. The CPP procedural timeline consisted of baseline preference testing (days 12-13 of the chronic regimen), conditioning (days 14-19, 2 daily 30-min sessions separated by 5 h), CPP test (day 21), extinction (days 22-34; no injections), CPP extinction test (day 35), and reinstatement test (day 36). Mice that had not extinguished received additional extinction sessions prior to reinstatement testing on day 42. On test days, mice were pretreated with either saline or CP94253 (10 mg/kg, IP). Testing began 30 min later, immediately after mice were primed with either saline or cocaine (5 mg/kg for locomotion; 15 mg/kg for reinstatement). We found that CP94253 increased spontaneous locomotion in mice receiving repeated injections of either saline or cocaine when tested 1 day after the last injection, but had no effect on spontaneous locomotion after 20 days abstinence from repeated injections. Surprisingly, cocaine-induced locomotion was sensitized regardless of whether the mice had received repeated saline or cocaine. CP94253 attenuated expression of the sensitized locomotion after 20 days abstinence. A control experiment in noninjected, drug-naïve mice showed that CP94253 had no effect on spontaneous or cocaine-induced locomotion. Mice reinstated cocaine-CPP when given a cocaine prime, and CP94253 pretreatment attenuated cocaine reinstatement.The findings suggest that stress from repeated saline injections and/or co-housing with cocaine-injected mice may cross-sensitize with cocaine effects on locomotion and that CP94253 attenuates these effects, as well as reinstatement of cocaine-CPP. This study supports the idea that 5-HT1BR agonists may be useful anti-cocaine medications.

Pharmacological evidence for an abstinence-induced switch in 5-HT1B receptor modulation of cocaine self-administration and cocaine-seeking behavior.

Studies examining serotonin-1B (5-HT1B) receptor manipulations on cocaine self-administration and cocaine-seeking behavior initially seemed discrepant. However, we recently suggested based on viral-mediated 5-HT1B-receptor gene transfer that the discrepancies are likely due to differences in the length of abstinence from cocaine prior to testing. To further validate our findings pharmacologically, we examined the effects of the selective 5-HT1B receptor agonist CP 94,253 (5.6 mg/kg, s.c.) on cocaine self-administration during maintenance and after a period of protracted abstinence with or without daily extinction training. We also examined agonist effects on cocaine-seeking behavior at different time points during abstinence. During maintenance, CP 94,253 shifted the cocaine self-administration dose-effect function on an FR5 schedule of reinforcement to the left, whereas following 21 days of abstinence CP 94,253 downshifted the function and also decreased responding on a progressive ratio schedule of reinforcement regardless of extinction history. CP 94,253 also attenuated cue-elicited and cocaine-primed drug-seeking behavior following 5 days, but not 1 day, of forced abstinence. The attenuating effects of CP 94,253 on the descending limb of the cocaine dose-effect function were blocked by the selective 5-HT1B receptor antagonist SB 224289 (5 mg/kg, i.p.) at both time points, indicating 5-HT1B receptor mediation. The results support a switch in 5-HT1B receptor modulation of cocaine reinforcement from facilitatory during self-administration maintenance to inhibitory during protracted abstinence. These findings suggest that the 5-HT1B receptor may be a novel target for developing medication for treating cocaine dependence.